C-reactive protein impairs immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS axis in multiple myeloma.

BACKGROUND: C-reactive protein (CRP) is a prototypical acute phase protein in humans with the function of regulating immune cells. Serum CRP levels are elevated in multiple myeloma (MM), associated with MM cell proliferation and bone destruction. However, its direct effects on T lymphocytes in MM have not been elucidated. METHODS: Public data sets were used to explore the correlation of CRP levels with immune cell infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro, repeated freeze-thaw myeloma cell lines were taken as tumor antigens to load dendritic cells (DCs) derived from HLA-A*0201-positive healthy donors. MM-specific cytotoxic T cells (MM-CTL) were obtained from T lymphocytes of the corresponding donors pulsed with these DCs. B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T cells were manipulated by transfecting with lentivirus encoding an anti-BCMA single-chain variable fragment. Then T cells from healthy controls, MM-CTLs and BCMA CAR-T cells were exposed to CRP and analyzed for cell proliferation, cytotoxicity, immunophenotypes. CRP binding capacity to T cells before and after Fc gamma receptors IIb (FcγRIIb) blockage, p38 mitogen-activated protein kinase (MAPK) pathway and the downstream molecules were also detected. In vivo, both normal C57BL/6J mice and the Vk*MYC myeloma mouse models were applied to confirm the impact of CRP on T cells. RESULTS: CRP levels were negatively correlated with cell-infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro experiments showed that CRP inhibited T-cell proliferation in a dose-dependent manner, impaired the cytotoxic activity and upregulated expression of senescent markers in CD8+ T cells. In vivo results validated the suppressive role of CRP in CD8+ T cells. CRP could bind to CD8+ T cells, mainly to the naïve T subset, while the binding was dramatically decreased by FcγRIIb blockage. Furthermore, CRP resulted in increased phosphorylation of p38 MAPK, elevated levels of reactive oxygen species and oxidized glutathione in CD8+ T cells. CONCLUSIONS: We found that CRP impaired immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS signaling pathway. The study casted new insights into the role of CRP in anti-myeloma immunity, providing implications for future immunotherapy in MM.

Unravelling the imbalanced Th17-like cell differentiation by single-cell RNA sequencing in multiple myeloma.

Multiple myeloma (MM) is a bone marrow resident hematological malignancy. T helper (Th) cells play an essential role in maladjustment of immune function and promotion of myeloma cell proliferation and survival, which has not been fully elucidated. Here, we compared transcriptome profiles of CD4+ T cells in bone marrow samples of 3 healthy individuals and 10 MM patients before and after treatment using single-cell RNA sequencing. CD4+ T cells were divided into 7 clusters. Imbalanced Th17-like cell differentiation was indicated in MM based on bioinformation analyses, which involved IL2-STAT5 pathways and transcription factors NKFB1, RELA, STAT3, and GTF2A2. Pseudotime trajectory analysis of CD4+ T cell clusters further uncovered the enhanced transition of Th17-like to regulatory T (Treg) cells in MM, which was featured by expression changes of PLAC8, NKFB1, RELA, STAT3, and STAT1 along with the developmental path. Reduced cell-cell interaction between MM cells and CD4+ naïve/recently activated naïve T cells via CD74-APP might lead to imbalanced Th17-like cell differentiation. Checkpoints via TIGIT-NECTIN3 and LGALS9-CD47 in Treg and MM cells were also identified. Our study reveals imbalanced differentiation pattern of Th17-like cells and the immunosuppressive profiles in connection with MM cells, which might help to shed light on CD4+ T cell function in MM.

Self-Aggregated Nanoscale Metal-Organic Framework for Targeted Pulmonary Decorporation of Uranium.

The limited availability of effective agents for removing actinides from the lungs significantly restricts the effectiveness of medical treatments for nuclear emergencies. Inhalation is the primary route of internal contamination in 44.3% of actinide-related accidents, leading to the accumulation of radionuclides in the lungs and resulting in infections and potential tumor formation (tumorigenesis). This study focuses on the synthesis of a nanometal-organic framework (nMOF) material called ZIF-71-COOH, which is achieved by post-synthetic carboxyl functionalization of ZIF-71. The material demonstrates high and selective adsorption of uranyl, while also exhibiting increased particle size (≈2100 nm) when it aggregates in the blood, enabling passive targeting of the lungs through mechanical filtration. This unique property facilitates the rapid enrichment and selective recognition of uranyl, making nano ZIF-71-COOH highly effective in removing uranyl from the lungs. The findings of this study highlight the potential of self-aggregated nMOFs as a promising drug delivery system for targeted uranium decorporation in the lungs.

Single-cell technologies in multiple myeloma: new insights into disease pathogenesis and translational implications.

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of plasma cells. Although therapeutic advances have been made to improve clinical outcomes and to prolong patients' survival in the past two decades, MM remains largely incurable. Single-cell sequencing (SCS) is a powerful method to dissect the cellular and molecular landscape at single-cell resolution, instead of providing averaged results. The application of single-cell technologies promises to address outstanding questions in myeloma biology and has revolutionized our understanding of the inter- and intra-tumor heterogeneity, tumor microenvironment, and mechanisms of therapeutic resistance in MM. In this review, we summarize the recently developed SCS methodologies and latest MM research progress achieved by single-cell profiling, including information regarding the cancer and immune cell landscapes, tumor heterogeneities, underlying mechanisms and biomarkers associated with therapeutic response and resistance. We also discuss future directions of applying transformative SCS approaches with contribution to clinical translation.

Single-cell RNA sequencing reveals XBP1-SLC38A2 axis as a metabolic regulator in cytotoxic T lymphocytes in multiple myeloma.

The mechanisms underlying the functional impairment and metabolic reprogramming of T lymphocytes in multiple myeloma (MM) have not been fully elucidated. In this study, single-cell RNA sequencing was used to compare gene expression profiles in T cells in bone marrow and peripheral blood of 10 newly diagnosed MM patients versus 3 healthy donors. Unbiased bioinformatics analysis revealed 9 cytotoxic T cell clusters. All 9 clusters in MM had higher expression of senescence markers (e.g., KLRG1 and CTSW) than the healthy control; some had higher expression of exhaustion-related markers (e.g., LAG3 and TNFRSF14). Pathway enrichment analyses showed downregulated amino acid metabolism and upregulated unfolded protein response (UPR) pathways, along with absent expression of glutamine transporter SLC38A2 and increased expression of UPR hallmark XBP1 in cytotoxic T cells in MM. In vitro studies revealed that XBP1 inhibited SLC38A2 by directly binding to its promoter, and silencing SLC38A2 resulted in decreased glutamine uptake and immune dysfunction of T cells. This study provided a landscape description of the immunosuppressive and metabolic features in T lymphocytes in MM, and suggested an important role of XBP1-SLC38A2 axis in T cell function.

Distinct mechanisms of dysfunctional antigen-presenting DCs and monocytes by single-cell sequencing in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy with the hallmark of immunodeficiency, including dysfunction of T cells, NK cells, and APCs. Dysfunctional APCs have been reported to play a key role in promoting MM progression. However, the molecular mechanisms remain elusive. Here, single-cell transcriptome analysis of dendritic cells (DC) and monocytes from 10 MM patients and three healthy volunteers was performed. Both DCs and monocytes were divided into five distinct clusters, respectively. Among them, monocyte-derived DCs (mono-DC) were shown to develop from intermediate monocytes (IM) via trajectory analysis. Functional analysis showed that, compared with healthy controls, conventional DC2 (cDC2), mono-DC, and IM of MM patients exhibited impaired antigen processing and presentation capacity. Moreover, reduced regulon activity of interferon regulatory factor 1 (IRF1) was found in cDC2, mono-DC and IM of MM patients according to single-cell regulatory network inference and clustering (SCENIC) analysis, while the downstream mechanisms were distinct. Specifically in MM patients, cathepsin S (CTSS) was markedly downregulated in cDC2, major histocompatibility complex (MHC) class II transactivator (CIITA) was significantly decreased in IM, in addition both CTSS and CIITA were downregulated in mono-DC based on differentially expressed genes analysis. In vitro study validated that knockdown of Irf1 downregulated Ctss and Ciita respectively in mouse DC cell line DC2.4 and mouse monocyte/macrophage cell line RAW264.7, which ultimately inhibited proliferation of CD4+ T cells after being cocultured with DC2.4 or RAW264.7 cells. This current study unveils the distinct mechanisms of cDC2, IM, and mono-DC function impairment in MM, offering new insight into the pathogenesis of immunodeficiency.

Dynamic single-cell RNA-seq analysis reveals distinct tumor program associated with microenvironmental remodeling and drug sensitivity in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of malignant plasma cells. Despite extensive research, molecular mechanisms in MM that drive drug sensitivity and clinic outcome remain elusive. RESULTS: Single-cell RNA sequencing was applied to study tumor heterogeneity and molecular dynamics in 10 MM individuals before and after 2 cycles of bortezomib-cyclophosphamide-dexamethasone (VCD) treatment, with 3 healthy volunteers as controls. We identified that unfolded protein response and metabolic-related program were decreased, whereas stress-associated and immune reactive programs were increased after 2 cycles of VCD treatment. Interestingly, low expression of the immune reactive program by tumor cells was associated with unfavorable drug response and poor survival in MM, which probably due to downregulation of MHC class I mediated antigen presentation and immune surveillance, and upregulation of markers related to immune escape. Furthermore, combined with immune cells profiling, we uncovered a link between tumor intrinsic immune reactive program and immunosuppressive phenotype in microenvironment, evidenced by exhausted states and expression of checkpoint molecules and suppressive genes in T cells, NK cells and monocytes. Notably, expression of YBX1 was associated with downregulation of immune activation signaling in myeloma and reduced immune cells infiltration, thereby contributed to poor prognosis. CONCLUSIONS: We dissected the tumor and immune reprogramming in MM during targeted therapy at the single-cell resolution, and identified a tumor program that integrated tumoral signaling and changes in immune microenvironment, which provided insights into understanding drug sensitivity in MM.

The epidemiology and prognosis of patients with primary gastric T-cell lymphoma in the SEER program.

BACKGROUND: Primary gastric T-cell lymphoma (PG-TCL) is a rare hematological malignancy with few data reported. The objective of this study is to investigate the epidemiology, clinical characteristics, and survivals of PG-TCL. METHODS: Totally, 164 patients with PG-TCL from 1975 to 2016 extracted from the Surveillance, Epidemiology, and End Results Program (SEER) database were analyzed. Kaplan-Meier method was applied to plot overall survival (OS) and cancer-specific survival (CSS). The prognostic factors of OS and CSS were explored by Cox proportional hazard regression. Nomograms were constructed to predict survival possibilities. RESULTS: The age-adjusted incidence rate of PG-TCL was 0.0091 per 100,000 person-years and increased with age. The median age at onset was 65 years old with male predominance. The major histological type was peripheral T-cell lymphoma, NOS (63.4%). The 1-, 2-, and 5-year OS were 45.5%, 34.7%, and 23.5%, respectively while the 1-, 2-, and 5-year CSS were 47.4%, 37.3%, and 29.6%, respectively. Multivariate Cox analysis demonstrated that age at diagnosis, use of chemotherapy, and radiotherapy were the independent prognostic factors for OS. Chemotherapy combined with radiotherapy could significantly improve patients' OS compared with chemotherapy alone. Moreover, age at diagnosis and use of chemotherapy were also the independent prognostic factors for CSS. Nomograms for PG-TCL were developed to predict 1-, 2-, and 5-year OS possibilities. The predictability of nomograms was verified by high concordance index and good agreement with the predicted value in calibration plots. CONCLUSION: PG-TCL is a rare neoplasm with low incidence. Patients with PG-TCL generally exhibited poor prognosis. Use of chemotherapy plus radiotherapy was associated with favorable OS.

Single-cell transcriptome profiling reveals the key role of ZNF683 in natural killer cell exhaustion in multiple myeloma.

BACKGROUNDS: Decreased cytotoxicity of natural killer (NK) cells has been shown in multiple myeloma (MM). However, the underlying molecular mechanisms remain unclear. Here, by using single-cell RNA sequencing analysis and in vitro experiments, we aim to uncover and validate molecularly distinctive insights into identifying regulators for NK cell exhaustion and provide potential targets for novel immune therapies in MM. METHODS: Single-cell RNA sequencing was conducted in the bone marrow and peripheral blood samples from 10 newly diagnosed MM patients and three healthy volunteers. Based on the cluster-defining differentially expressed genes, we named and estimated functional states of each cluster via bioinformatics analyses. Functional significance of key findings obtained from sequencing analysis was examined in a series of in vitro experiments, including luciferase reporter assay, lentiviral expression vector construction, NK cell transfection, RT-qPCR, flow cytometry, and cytotoxicity assay. RESULTS: We classified NK cells into seven distinct clusters and confirmed that a subset of ZNF683+ NK cells were enriched in MM patients with 'exhausted' transcriptomic profile, featuring as decreased expression of activating receptors and cytolytic molecules, as well as increased expression of inhibitory receptors. Next, we found a significant downregulation of SH2D1B gene that encodes EAT-2, an adaptor protein of activating receptor SLAMF7, in ZNF683+ NK cells from MM patients versus healthy volunteers. We further proved that ZNF683 transfection in NK cells significantly downregulated SH2D1B expression via directly binding to the promoter of SH2D1B, leading to NK cell cytotoxic activity impairment and exhausted phenotypes acquisition. In contrast, ZNF683 knockout in NK cells from MM patients increased cytotoxic activity and reversed NK cell exhaustion. CONCLUSIONS: In summary, our findings uncover an important mechanism of ZNF683+ NK cell exhaustion and suggest that transcriptional suppressor ZNF683 as a potential useful therapeutic target in immunotherapy of MM.

Identification of Immune-Related Genes for Risk Stratification in Multiple Myeloma Based on Whole Bone Marrow Gene Expression Profiling.

Background: Multiple myeloma (MM) is characterized by abnormal proliferation of bone marrow clonal plasma cells. Tumor immunotherapy, a new therapy that has emerged in recent years, offers hope to patients, and studying the expression characteristics of immune-related genes (IRGs) based on whole bone marrow gene expression profiling (GEP) in MM patients can help guide personalized immunotherapy. Methods: In this study, we explored the potential prognostic value of IRGs in MM by combining GEP and clinical data from the GEO database. We identified hub IRGs and transcription factors (TFs) associated with disease progression by Weighted Gene Co-expression Network Analysis (WGCNA), and modeled immune-related prognostic signature by univariate and multivariate Cox and least absolute shrinkage and selection operator (LASSO) regression analysis. Subsequently, the prognostic ability of signature was verified by multiple statistical methods. Moreover, ssGSEA and GSEA algorithm reveled different immunological characteristics and biological function variation in different risk groups. We mapped the hub IRGs by protein-protein interaction network (PPI) and extracted the top 10 ranked genes. Finally, we conducted vitro assays on two alternative IRGs. Results: Our study identified a total of 14 TFs and 88 IRGs associated with International Staging System (ISS). Ten IRGs were identified by Cox -LASSO regression analysis, and used to develop optimal prognostic signature for overall survival (OS) in MM patients. The 10-IRGs were BDNF, CETP, CD70, LMBR, LTBP1, NENF, NR1D1, NR1H2, PTK2B and SEMA4. In different groups, risk signatures showed excellent survival prediction ability, and MM patients also could be stratified at survival risk. In addition, IRF7 and SHC1 were hub IRGs in PPI network, and the vitro assays proved that they could promote tumor progression. Notably, ssGSEA and GSEA results confirmed that different risk groups could accurately indicate the status of tumor microenvironment (TME) and activation of biological pathways. Conclusion: Our study suggested that immune-related signature could be used as prognostic markers in MM patients.

Increased Expression of SETDB1 Predicts Poor Prognosis in Multiple Myeloma.

Several genes on chromosome 1q21 region predict a high risk of multiple myeloma (MM); however, the underlying molecular pathology remains elusive. Overexpression, amplification, or activation of SET Domain Bifurcated 1 (SETDB1), which is located on 1q21, is closely associated with poor prognosis of many human solid malignancies. In our study, upregulation of SETDB1 might indicate an unfavorable prognosis of MM using bioinformatics analysis from GEO databases and MMRF-CoMMpass. Here, increased SETDB1 expression was observed in the plasma cells from newly diagnosed multiple myeloma patients compared to those from the normal controls. Meanwhile, SETDB1 overexpression was the result of increased copy numbers of SETDB1 gene. In MM patients, the Kaplan-Meier analysis was employed to demonstrate that increased SETDB1 expression was associated with shorter overall survival (OS) and event-free survival (EFS). Besides, we conducted multifactorial cox regression analysis to state that SETDB1 expression was an independent biomarker for OS and EFS. MM patients with higher SETDB1 expression showed higher levels of beta-2 microglobulin (ß2M), lactate dehydrogenase (LDH), and bone marrow biopsy plasma cells (BMPC) and lower levels of haemoglobin (HGB). Functional enrichment analysis suggested that SETDB1 could promote cell cycle progression in myeloma. Finally, we observed that SETDB1 was distinctly correlated with tumor immunity in MM. SETDB1 expression in myeloma cells was positively correlated with CD56dim natural killer cells but negatively correlated with infiltrating levels of type17 T helper cells, effector memory CD8 T cells, activated dendritic cells, and natural killer T cells from whole bone marrow (WBM) biopsies. Taken together, these results indicated that SETDB1 could be used as a novel biomarker for predicting the prognosis of MM patients.

A Five-Gene Risk Score Model for Predicting the Prognosis of Multiple Myeloma Patients Based on Gene Expression Profiles.

Multiple myeloma is a heterogeneous plasma cell malignancy that remains incurable because of the tendency of relapse for most patients. Survival outcomes may vary widely due to patient and disease variables; therefore, it is necessary to establish a more accurate prognostic model to improve prognostic precision and guide clinical therapy. Here, we developed a risk score model based on myeloma gene expression profiles from three independent datasets: GSE6477, GSE13591, and GSE24080. In this model, highly survival-associated five genes, including EPAS1, ERC2, PRC1, CSGALNACT1, and CCND1, are selected by using the least absolute shrinkage and selection operator (Lasso) regression and univariate and multivariate Cox regression analyses. At last, we analyzed three validation datasets (including GSE2658, GSE136337, and MMRF datasets) to examine the prognostic efficacy of this model by dividing patients into high-risk and low-risk groups based on the median risk score. The results indicated that the survival of patients in low-risk group was greatly prolonged compared with their counterparts in the high-risk group. Therefore, the five-gene risk score model could increase the accuracy of risk stratification and provide effective prediction for the prognosis of patients and instruction for individualized clinical treatment.

A breeding strategy targeting the secondary gene pool of bread wheat: introgression from a synthetic hexaploid wheat.

KEY MESSAGE: Introgressing one-eighth of synthetic hexaploid wheat genome through a double top-cross plus a two-phase selection is an effective strategy to develop high-yielding wheat varieties. The continued expansion of the world population and the likely onset of climate change combine to form a major crop breeding challenge. Genetic advances in most crop species to date have largely relied on recombination and reassortment within a relatively narrow gene pool. Here, we demonstrate an efficient wheat breeding strategy for improving yield potentials by introgression of multiple genomic regions of de novo synthesized wheat. The method relies on an initial double top-cross (DTC), in which one parent is synthetic hexaploid wheat (SHW), followed by a two-phase selection procedure. A genotypic analysis of three varieties (Shumai 580, Shumai 969 and Shumai 830) released from this program showed that each harbors a unique set of genomic regions inherited from the SHW parent. The first two varieties were generated from very small populations, whereas the third used a more conventional scale of selection since one of bread wheat parents was a pre-breeding material. The three varieties had remarkably enhanced yield potential compared to those developed by conventional breeding. A widely accepted consensus among crop breeders holds that introducing unadapted germplasm, such as landraces, as parents into a breeding program is a risky proposition, since the size of the breeding population required to overcome linkage drag becomes too daunting. However, the success of the proposed DTC strategy has demonstrated that novel variation harbored by SHWs can be accessed in a straightforward, effective manner. The strategy is in principle generalizable to any allopolyploid crop species where the identity of the progenitor species is known.

Humanin analog enhances the protective effect of dexrazoxane against doxorubicin-induced cardiotoxicity.

The chemotherapeutic effect of doxorubicin (Dox) is limited by cumulative dose-dependent cardiotoxicity in cancer survivors. Dexrazoxane (DRZ) is approved to prevent Dox-induced cardiotoxicity. Humanin and its synthetic analog HNG have a cytoprotective effect on the heart. To investigate the cardioprotective efficacy of HNG alone or in combination with DRZ against Dox-induced cardiotoxicity, 80 adult male mice were randomly divided into 8 groups to receive the following treatments via intraperitoneal injection: saline dailym HNG (5 mg/kg) daily, DRZ (60 mg/kg) weekly, Dox (3 mg/kg) weekly, DRZ + HNG, Dox + HNG, Dox + DRZ, and Dox + HNG + DRZ. Echocardiograms were performed before and at 4, 8, and 9.5 wk after the beginning of treatment. All mice were euthanized at 10 wk. In the absence of Dox, HNG, DRZ, or DRZ + HNG had no adverse effect on the heart. Dox treatment caused decreases in ejection fraction and cardiac mass and increases in cardiomyocyte apoptosis and intracardiac fibrosis. HNG or DRZ alone blunted the Dox-induced decrease in left ventricle posterior wall thickness and modestly ameliorated the Dox-induced decrease in ejection fraction. HNG + DRZ significantly ameliorated Dox-induced decreases in ejection function, cardiac fibrosis, and cardiac mass. Using a targeted analysis for the mitochondrial gene array and protein expression in heart tissues, we demonstrated that HNG + DRZ reversed DOX-induced altered transcripts that were biomarkers of cardiac damage and uncoupling protein-2. We conclude that HNG enhances the cardiac protective effect of DRZ against Dox-induced cardiotoxicity. HNG + DRZ protects mitochondria from Dox-induced cardiac damage and blunts the onset of cardiac dysfunction. Thus, HNG may be an adjuvant to DRZ in preventing Dox-induced cardiotoxicity. NEW & NOTEWORTHY Doxorubicin (Dox) is commonly used for treating a wide range of human cancers. However, cumulative dosage-dependent carditoxicity often limits its clinical applications. We demonstrated in this study that treating young adult male mice with synthetic humanin analog enhanced the cardiac protective effect of dexrazoxane against chemotherapeutic agent Dox-induced cardiac dysfunction. Thus, humanin analog can potentially serve as an adjuvant to dexrazoxane in more effectively preventing Dox-induced cardiac dysfunction and cardiomyopathy.

NOD2/CARD15 gene mutation identified in a Chinese family with Blau syndrome.

PURPOSE: To characterize the clinical features of a Chinese pedigree with Blau syndrome and to identify mutations in the NOD2/CARD15 (nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family, member 15) gene. METHODS: Clinical features of this family were evaluated. Genomic DNA was obtained from blood samples, and all exons of NOD2/CARD15 were amplified by polymerase chain reaction (PCR) and direct DNA sequencing of PCR products was performed for mutations in NOD2/CARD15. RESULTS: Granulomatous arthritis, uveitis, and skin granulomas were found in all affected members. Sequencing analysis demonstrated a heterozygous C>T mutation in exon 4 of NOD2/CARD15 in all patients of this pedigree, which resulted in an amino acid substitution at position 334 (p.R334W). CONCLUSIONS: The R334W mutation in NOD2/CARD15 caused Blau syndrome in a Chinese pedigree. This is the first report of R334W mutation in NOD2/CARD15 in a Chinese pedigree of this disease.